Legumain is a protein that in human is encoded by the LGMN gene (Chen J. M. et al., J. Biol. Chem. 1997; Tanaka T. et al., Cytogenet. Cell Genet. 1966; Halfon S. et al., FEBS Lett. 1998). This gene encodes a cysteine proteinase that has a strict specificity for hydrolysis of asparaginyl bonds, cleaving peptide bonds with Asn or Asp at the P1 position (Schwarz G. et al., J. Biol. Chem. 2002). Several alternatively spliced transcript variants have been described, but the biological validity of only two have been determined. These two variants encode the same isoform (Yamane T. et al., Biochem. Biophys. Res. Commum. 2013). Legumain is over expressed in colorectal cancer, ovarian cancer, breast cancer and other types of cancers (Liu C. et al., Cancer Res. 2003; Murthy R. V. et al., Clin. Cancer Res. 2005). Legumain was identified as a tumor biomarker from gene expression profiling and tumor tissue array (Guo P. et al., PLoS One, 2013). Mammalian legumain is involved in the processing of bacterial peptides and endogenous proteins for MHC class II presentation in the lysosomal/endsommal systems. Legumain has been observed to be a potential predictive biomarker for several types of cancers (Wu M. et al., Asian Pac. J. Cancer Prev. 2014; Gawenda J. et al., Breast Cancer Res. Treat. 2007). It was demonstrated in membrane-associated vesicles concentrated at the invadopodia of tumor cells and on cell surfaces where it co-localized with integrins (Liu Y., Mol. Pharm. 2012). Overexpression of legumain may be associated with the formation of solid tumors (Liu et al., Cancer Res. 2003). Cells overexpressing legumain can possess increased migratory and invasive activity in vitro and adopt an invasive and metastatic phenotype in vivo, inferring the significance of legumain in tumor invasion and metastasis (Lin Y. et al., J. Nat. Cancer Inst. 2014). It has also been demonstrated that legumain promotes cell migration, and overexpression is associated with enhanced tissue invasion and metastasis (Haugen et al., Eur. J. Cancer 2015). The unique functional properties of legumain and its high-level expression in many human tumors support that it is a potential candidate as enzymatic target for prodrug activation and tumor eradicative therapy (Cheng Liu et al., Cancer Res. 2003). A prodrug strategy incorporating a legumain cleavable peptide substrate onto doxorubicin was developed (Liu et al., Nat Commun. 2014; Stern et al., Bioconjug. Chem. 2009). Despite that legumain is attractive as a target for anticancer drug design and the progress of pharmaceutical research on this target, there is a lack of clinical diagnostic method for determination of blood levels of legumain in an automated fashion by clinical chemistry analyzers or automated immunoanalyzers. Therefore, it is desired to develop a fully automated legumain test that is adaptable to clinical chemistry analyzers or immunoanalyzers in clinical settings.